Scottish Collaboration On Translational Research into Renal Cell Cancer SCOTRRCC

Scotland, United Kingdom

Scottish Collaboration On Translational Research into Renal Cell Cancer SCOTRRCC

Scotland, United Kingdom
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Lubbock A.L.R.,University of Edinburgh | Lubbock A.L.R.,Vanderbilt University | Stewart G.D.,University of Edinburgh | Stewart G.D.,Scottish Collaboration On Translational Research into Renal Cell Cancer SCOTRRCC | And 12 more authors.
BMC Medicine | Year: 2017

Background: Metastatic clear cell renal cell cancer (mccRCC) portends a poor prognosis and urgently requires better clinical tools for prognostication as well as for prediction of response to treatment. Considerable investment in molecular risk stratification has sought to overcome the performance ceiling encountered by methods restricted to traditional clinical parameters. However, replication of results has proven challenging, and intratumoural heterogeneity (ITH) may confound attempts at tissue-based stratification. Methods: We investigated the influence of confounding ITH on the performance of a novel molecular prognostic model, enabled by pathologist-guided multiregion sampling (n = 183) of geographically separated mccRCC cohorts from the SuMR trial (development, n = 22) and the SCOTRRCC study (validation, n = 22). Tumour protein levels quantified by reverse phase protein array (RPPA) were investigated alongside clinical variables. Regularised wrapper selection identified features for Cox multivariate analysis with overall survival as the primary endpoint. Results: The optimal subset of variables in the final stratification model consisted of N-cadherin, EPCAM, Age, mTOR (NEAT). Risk groups from NEAT had a markedly different prognosis in the validation cohort (log-rank p = 7.62 × 10-7; hazard ratio (HR) 37.9, 95% confidence interval 4.1-353.8) and 2-year survival rates (accuracy = 82%, Matthews correlation coefficient = 0.62). Comparisons with established clinico-pathological scores suggest favourable performance for NEAT (Net reclassification improvement 7.1% vs International Metastatic Database Consortium score, 25.4% vs Memorial Sloan Kettering Cancer Center score). Limitations include the relatively small cohorts and associated wide confidence intervals on predictive performance. Our multiregion sampling approach enabled investigation of NEAT validation when limiting the number of samples analysed per tumour, which significantly degraded performance. Indeed, sample selection could change risk group assignment for 64% of patients, and prognostication with one sample per patient performed only slightly better than random expectation (median logHR = 0.109). Low grade tissue was associated with 3.5-fold greater variation in predicted risk than high grade (p = 0.044). Conclusions: This case study in mccRCC quantitatively demonstrates the critical importance of tumour sampling for the success of molecular biomarker studies research where ITH is a factor. The NEAT model shows promise for mccRCC prognostication and warrants follow-up in larger cohorts. Our work evidences actionable parameters to guide sample collection (tumour coverage, size, grade) to inform the development of reproducible molecular risk stratification methods. © 2017 The Author(s).


Stewart G.D.,University of Edinburgh | Stewart G.D.,Scottish Collaboration On Translational Research into Renal Cell Cancer SCOTRRCC | Stewart G.D.,University of Cambridge | Powles T.,The Royal Free Hospital | And 14 more authors.
Oncotarget | Year: 2016

Background: Genetic intratumoral heterogeneity (ITH) hinders biomarker development in metastatic clear cell renal cancer (mccRCC). Epigenetic relative to genetic ITH or the presence of consistent epigenetic changes following targeted therapy in mccRCC have not been evaluated. The aim of this study was to determine methylome/genetic ITH and to evaluate specific epigenetic and genetic changes associated with sunitinib therapy. Patients and methods: Multi-region DNA sampling performed on sequential frozen pairs of primary tumor tissue from 14 metastatic ccRCC patients, in the Upfront Sunitinib (SU011248) Therapy Followed by Surgery in Patients with Metastatic Renal Cancer: a Pilot Phase II Study (SuMR; ClinicalTrials.gov identifier: NCT01024205), at presentation (biopsy) and after 3-cycles of 50mg sunitinib (nephrectomy). Untreated biopsy and nephrectomy samples before and after renal artery ligation were controls. Ion Proton sequencing of 48 key ccRCC genes, and MethylCap-seq DNA methylation analysis was performed, data was analysed using the statistical computing environment R. Results: Unsupervised hierarchical clustering revealed complete methylome clustering of biopsy and three nephrectomy samples for each patient (14/14 patients). For mutational status, untreated biopsy and all treated nephrectomy samples clustered together in 8/13 (61.5%) patients. The only methylation target significantly altered following sunitinib therapy was VHL promoter region 7896829 which was hypermethylated with treatment (FDR=0.077, P<0.001) and consistent for all patients (pre-treatment 50% patients had VHL mutations, 14% patients VHL hypermethylation). Renal artery ligation did not affect this result. No significant differences in driver or private mutation count was found with sunitinib treatment. Conclusions: Demonstration of relative methylome homogeneity and consistent VHL hypermethylation, after sunitinib, may overcome the hurdle of ITH present at other molecular levels for biomarker research.


Stewart G.D.,University of Edinburgh | Stewart G.D.,Scottish Collaboration on Translational Research into Renal Cell Cancer SCOTRRCC | O'Mahony F.C.,University of Edinburgh | O'Mahony F.C.,Scottish Collaboration on Translational Research into Renal Cell Cancer SCOTRRCC | And 23 more authors.
Clinical Cancer Research | Year: 2015

Purpose: The aim of this study was to investigate the effect of VEGF-targeted therapy (sunitinib) on molecular intratumoral heterogeneity (ITH) in metastatic clear cell renal cancer (mccRCC). Experimental Design: Multiple tumor samples (n = 187 samples) were taken from the primary renal tumors of patients with mccRCC who were sunitinib treated (n = 23, SuMR clinical trial) or untreated (n = 23, SCOTRRCC study). ITH of pathologic grade, DNA (aCGH), mRNA (Illumina Beadarray) and candidate proteins (reverse phase protein array) were evaluated using unsupervised and supervised analyses (driver mutations, hypoxia, and stromal-related genes). ITH was analyzed using intratumoral protein variance distributions and distribution of individual patient aCGH and gene-expression clustering. Results: Tumor grade heterogeneity was greater in treated compared with untreated tumors (P = 0.002). In unsupervised analysis, sunitinib therapy was not associated with increased ITH in DNA or mRNA. However, there was an increase in ITH for the driver mutation gene signature (DNA and mRNA) as well as increasing variability of protein expression with treatment (P < 0.05). Despite this variability, significant chromosomal and transcript changes to key targets of sunitinib, such as VHL, PBRM1, and CAIX, occurred in the treated samples. Conclusions: These findings suggest that sunitinib treatment has significant effects on the expression and ITH of key tumor and treatment specific genes/proteins in mccRCC. The results, based on primary tumor analysis, do not support the hypothesis that resistant clones are selected and predominate following targeted therapy. © 2015 AACR.


PubMed | Scottish Collaboration On Translational Research into Renal Cell Cancer SCOTRRCC, Ghent University, The Royal Free Hospital and University of Edinburgh
Type: Journal Article | Journal: Oncotarget | Year: 2016

Genetic intratumoral heterogeneity (ITH) hinders biomarker development in metastatic clear cell renal cancer (mccRCC). Epigenetic relative to genetic ITH or the presence of consistent epigenetic changes following targeted therapy in mccRCC have not been evaluated. The aim of this study was to determine methylome/genetic ITH and to evaluate specific epigenetic and genetic changes associated with sunitinib therapy.Multi-region DNA sampling performed on sequential frozen pairs of primary tumor tissue from 14 metastatic ccRCC patients, in the Upfront Sunitinib (SU011248) Therapy Followed by Surgery in Patients with Metastatic Renal Cancer: a Pilot Phase II Study (SuMR; ClinicalTrials.gov identifier: NCT01024205), at presentation (biopsy) and after 3-cycles of 50mg sunitinib (nephrectomy). Untreated biopsy and nephrectomy samples before and after renal artery ligation were controls. Ion Proton sequencing of 48 key ccRCC genes, and MethylCap-seq DNA methylation analysis was performed, data was analysed using the statistical computing environment R.Unsupervised hierarchical clustering revealed complete methylome clustering of biopsy and three nephrectomy samples for each patient (14/14 patients). For mutational status, untreated biopsy and all treated nephrectomy samples clustered together in 8/13 (61.5%) patients. The only methylation target significantly altered following sunitinib therapy was VHL promoter region 7896829 which was hypermethylated with treatment (FDR=0.077, P<0.001) and consistent for all patients (pre-treatment 50% patients had VHL mutations, 14% patients VHL hypermethylation). Renal artery ligation did not affect this result. No significant differences in driver or private mutation count was found with sunitinib treatment.Demonstration of relative methylome homogeneity and consistent VHL hypermethylation, after sunitinib, may overcome the hurdle of ITH present at other molecular levels for biomarker research.

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